大豆异黄酮代谢终产物——雌马酚的研究进展

雌马酚是大豆异黄酮在人体肠道中的最终代谢产物。众多研究表明大豆异黄酮具有广泛的保健价值,在预防心血管疾病、乳腺癌和前列腺癌等方面均发挥重要作用。大豆异黄酮的生物活性在某种程度上归因于雌马酚,并且雌马酚具有较其前体更高的生物学效用。大豆异黄酮在特定的肠道菌作用下降解产生雌马酚,但并非每个人体内都可代谢产生雌马酚。总结了雌马酚在人体中的生理活性,并且综合论述了化学合成制备混合型雌马酚的方法,以及生物合成雌马酚过程中,动物及人体内可降解大豆异黄酮的肠道菌筛选情况。     

肠道菌株单独及复合培养产雌马酚能力的评价

以常见的五种益生菌乳酸杆菌、乳酸芽孢杆菌、双歧杆菌、嗜热链球菌、枯草芽孢杆菌和来自素食者粪便分离得到的两种菌LJ-G_1和LJ-Q_2共7种肠道菌为受试菌,培养中添加大豆异黄酮,采用紫外分光光度法对培养基中的雌马酚进行定性定量分析,评价7种肠道菌雌马酚转化力。然后将高产雌马酚的菌按一定比例复合考察协同作用,再进一步探讨复合菌适宜培养条件。结果表明:乳酸杆菌、嗜热链球菌、LJ-G_1、LJ-Q_2均有产雌马酚的能力,而且素食者肠道菌LJ-G_1、LJ-Q_2产雌马酚能力较高,分别为15.92μg/mL和14.43μg/mL;菌株LJ-G-1、LJ-Q_2、乳酸杆菌三者复合产雌马酚能力有协同作用,在19.04~20.78μg/mL之间。选取LJ-G1与LJ-Q2复合培养,适宜复合比例为1∶1、培养基初始pH7.0、培养温度37℃,雌马酚转化力为21.23μg/mg。自肠道分离菌代谢大豆异黄酮产雌马酚能力较高。     

大豆食品对人体内异黄酮代谢水平影响的研究

探讨不同苷元含量的大豆食品的摄入对人体内异黄酮代谢水平的影响。应用现状调查方法,筛选出86名居住在哈尔滨市区健康成年男女。采用高压液相色谱法分析摄入大豆异黄酮前后尿中雌马酚等代谢物24 h排泄量,计算出产雌马酚者比例。然后从产雌马酚者中随机选取12名受试者,在3个时期分别摄入豆浆、β-葡糖苷酶处理的豆浆和豆腐。在摄入后0～48 h收集尿液。通过高压液相色谱法测定尿液中大豆苷元、染料木素和雌马酚的含量以确定糖苷或苷元的摄入是否影响异黄酮的代谢。结果是尿液中大豆苷元、染料木素和雌马酚的排泄量差异不显著。结论是人体内异黄酮的代谢可能不受大豆食品中异黄酮苷元含量的影响。     

1 株素食者产雌马酚肠道菌的分离与鉴定

以素食者粪便为样品,大豆异黄酮为底物,对产雌马酚的肠道菌进行分离培养及鉴定。采用紫外光谱法对雌马酚定性、定量测定,用肉眼观察、扫描电镜、生理生化、16S rDNA全序测序方法对分离得到的LJ-G1肠道菌进行菌株鉴定。结果表明：麦康凯和BHI培养基、厌氧平板划线培养法更适于产雌马酚肠道菌的分离筛选。采取10 g粪样稀释到10-5接种,在pH 7.3、温度37 ℃、厌氧培养36 h的条件下,培养基中雌马酚产出量达到10 μg/mL以上。经鉴定分离得到的LJ-G1菌株为G－杆菌;根据生理生化反应结果,查阅《常见细菌系统鉴定手册》,确定LJ-G1为河生肠杆菌;16S rDNA全序测序显示与肠杆菌同源性达99%。     

雌马酚制备、检测和富集技术研究进展

雌马酚（equol,Eq）是大豆异黄酮的代谢产物之一,具有较高生物活性和稳定化学结构,其生物活性远远高于大豆异黄酮,且易被人体肠道吸收利用。然而目前仅有30%～60%的人群可以通过自身代谢产生雌马酚,而其他一部分人群则需外源物质的补充来满足其对雌马酚的需求。因此,如何高效、快速地制备并检测雌马酚已经成为食品、生物和医药等多个领域的研究热点。该文对雌马酚的制备、检测以及发酵富集技术等方面的研究进行总结,旨在为雌马酚在食品领域的进一步开发研究提供一定的参考。     

高效液相色谱法测定六种大豆异黄酮及雌马酚的含量

针对豆制品中同时测定大豆异黄酮和雌马酚方法存在的问题,优化并建立了同时测定6种大豆异黄酮和雌马酚的高效液相检测程序,并测定了几种市售豆乳中大豆异黄酮的含量。采用乙腈/甲醇（v/v,5/2）和水为流动相、流速1.0mL/min、检测波长254nm,6种大豆异黄酮和雌马酚均得到良好的分离,相关系数R2分别为0.9998、0.9989、0.9997、0.9986、0.9998、0.9995、0.9999,线性关系良好。该方法准确、灵敏,适合于豆制品中大豆异黄酮及雌马酚的检测。     

雌马酚及大豆活性成分对人前列腺癌细胞DU145的生长影响

目的:观察大豆异黄酮代谢产物雌马酚对人前列腺癌细胞DU145的生长影响,并在生理浓度下比较其与大豆异黄酮主要成分金雀异黄素、大豆素的体外抗增殖活性。方法:采用MTT法检测不同浓度的雌马酚、金雀异黄素及大豆素对DU145细胞增殖的影响,并以流式细胞术分析细胞周期分布情况。结果:雌马酚在1×10-6～1×10-5mol/L表现为抑制作用,金雀异黄素在1×10-7～1×10-6mol/L时表现为促增殖效应,达到5×10-5mol/L时出现抑制作用,大豆素在生理浓度下则主要表现为促增殖效应;雌马酚在1×10-6mol/L时使细胞在G0/G1期阻滞,在5×10-6～1×10-5mol/L时使S期及G2/M期细胞增多,金雀异黄素在5×10-6mol/L出现G2/M期阻滞,大豆素使S期及G2/M期细胞增多。结论:雌马酚在体外具有一定的抗前列腺癌活性;在生理浓度下,其抗癌活性可能强于金雀异黄素及大豆素。     

大豆异黄酮降解液中雌马酚纯化工艺

为从大豆异黄酮降解液中分离纯化雌马酚,以雌马酚的含量为指标,确定聚酰胺树脂为吸附剂纯化雌马酚。通过单因素试验和正交试验,确定最佳吸附条件为粗提液中雌马酚质量浓度2.40μg/mL、pH5、吸附时间6h,得雌马酚吸附率45.36%,最佳解吸条件为乙醇溶液体积分数80%、乙醇溶液用量45mL、乙醇溶液流速0.5mL/min,得解吸率60%。     

臭豆腐中雌马酚的测定及其影响因素

目的:研究臭豆腐中雌马酚的含量及其影响因素,以期为选择良好食物性来源的雌马酚提供理论依据。方法:采用反相-高效液相色谱法(RP-HPLC)检测我国市场上常见的4个品牌臭豆腐中雌马酚的含量(对比其它8种发酵豆制品)及其与大豆异黄酮含量和理化指标等因素的Spearnam相关性,并对卤水及其浸泡和油炸时长沙HSJD臭豆腐中雌马酚的含量进行分析。结果:4种臭豆腐中均含雌马酚,其它发酵豆制品均不含雌马酚。臭豆腐中雌马酚的含量在11.93～24.33μg/g干物质范围,其中,长沙HSJD臭豆腐中的含量最高(24.33μg/g干物质)。雌马酚含量与大豆苷元、染料木苷元含量呈显著正相关关系(P<0.05),相关系数分别为0.335,0.411;与氯化物、氨基酸态氮、总酸含量呈极显著负相关关系(P<0.01),相关系数分别为-0.463,-0.506,-0.674;与p H值呈极显著正相关关系(P<0.01),相关系数为0.539。添加豆腐脑发酵一年(STB1)和发酵二年(STB2)的长沙HSJD臭豆腐卤水中均含雌马酚,含量分别为168.31,309.62μg/g干物质,而未加豆腐脑的...     

大豆异黄酮生理活性的研究进展——(1)大豆异黄酮的代谢及雌激素特性

大豆尤其是大豆胚轴富含有益于人类健康的大豆异黄酮。人体内的两种专一肠道细菌能将大豆异黄酮中的大豆苷和染料木苷代谢为相应的糖苷配基大豆黄素和染料木黄酮。马雌酚是大豆黄素在肠内细菌作用下的代谢产物,具有比大豆异黄酮更高的生理活性,但是并非在所有摄取大豆异黄酮的健康成年人体内都能产生马雌酚,马雌酚的产生与体内是否存在某些特定的肠内菌丛有关。大豆蛋白能影响肠内菌丛的构成,进而提高大豆异黄酮的生物利用率。大豆异黄酮是一类选择性雌激素受体调节剂,具有雌激素和抗雌激素的双重活性,可安全有效地用于预防和治疗妇女更年期综合症。     

Metabolism of dietary soy isoflavones to equol by human intestinal microflora--implications for health

Soy isoflavones have received considerable attention. Individuals with isoflavones-rich diets have significantly lower occurrences of cardiovascular disease, osteoporosis, and some cancers. The clinical effectiveness of soy isoflavones may be a function of the ability to biotransform soy isoflavones to the more potent estrogenic metabolite, equol, which may enhance the actions of soy isoflavones, owing to its greater affinity for estrogen receptors, unique antiandrogenic properties, and superior antioxidant activity. However, not all individuals consuming daidzein produce equol. Only approximately one-third to one-half of the population is able to metabolize daidzein to equol. This high variability in equol production is presumably attributable to interindividual differences in the composition of the intestinal microflora, which may play an important role in the mechanisms of action of isoflavones. But, the specific bacterial species in the colon involved in the production of equol are yet to be discovered. Therefore, future researches are aimed at identifying the specific bacterial species and strains that are capable of converting daidzein to equol or increasing equol production.     

素食者产雌马酚肠道菌生长条件优化

以菌落数和光密度值以及雌马酚含量为评价指标,测定两株肠道菌的生长曲线,考察金属盐MnSO4、ZnSO4、CaCl2、Na2CO3、KNO3对其生长状况的影响,筛查其对不同碳源（葡萄糖、麦芽糖、蔗糖、果糖、乳糖）的利用情况。其次采用单因素试验和正交试验对生长条件进行优化。结果表明：LJ-G1 和LJ-Q2两株菌的生长规律为在37 ℃厌氧条件下,培养6 h后进入指数期、21 h后进入生长平稳期、39 h后为衰亡期。金属盐对菌株生长均有抑制作用,作用最强的为MnSO4;葡萄糖对肠道菌的生长有促进作用。生长最优条件为：培养基中葡萄糖质量浓度7 g/L、pH 7.2、NaCl质量浓度2 g/L,雌马酚的产量较高为11.89 μg/mL。     

A new biotechnological process to enhance the soymilk bioactivity

Equol, a daidzein metabolite produced exclusively by intestinal bacteria in some, but not all, humans, exhibits a wide range of beneficial health effects owing to its superior nutraceutical effect compared with isoflavones of soy. The aim of this work was to develop bioprocesses capable of increasing the bioactive properties of soymilk and, most importantly, increase the equol content by a biotechnological process in vitro. Biotransformation processes based on soymilk fermentation by probiotic lactic bacteria and application of the enzyme tannase caused an increase in the bioactive isoflavones and antioxidant capacity of soymilk. Furthermore, these processes approximately resulted in a 10-fold increase in the equol content of the soymilk. This is the first study to produce a significant equol concentration in soymilk using enzymatic processing only. The results suggest a new and effective biotechnological process, with major commercial potential, capable of producing a bioactive soy extract that intends to be “functional for everyone.”     

Determination of in vitro isoflavone degradation in rumen fluid

The aim of this study was to determine the degradation of dietary isoflavones in rumen fluid under 2 feeding regimens. The experiments were performed in vitro using a rumen fluid buffer system. The rumen fluid was taken from cows fed either a hay diet or a concentrate-rich diet (the diet consisted of 34.6% maize silage, 17.6% haylage, 12.8% alfalfa hay, and 35.0% supplemental mixture on a dry matter basis). As a source of isoflavones, 40% soybean extract (Biomedica, Prague, Czech Republic) at levels of 5, 25, 50, and 75 mg per 40 mL of rumen fluid was used. Samples of soybean extract were incubated in triplicate at 39°C for 0, 3.0, 6.0, 12.0, and 24.0 h in incubation solution. The metabolism of daidzein and genistein was faster under concentrate-rich diet conditions. In general, production of equol started after 3 to 6 h of incubation and reached the highest rate after approximately 12 h of incubation regardless of the type of diet or concentration of extract. In most of the experiments, production of equol continued after 24 h of incubation. Generally, equol production was greater under the hay diet conditions. Furthermore, experiments with higher amounts of added soybean extract revealed possible inhibitory effects of high levels of isoflavones on the rumen microflora.     

Nutrikinetics of Isoflavone Metabolites After Fermented Soybean Product (Cheonggukjang) Ingestion in Ovariectomized Mice

1 Scope

Cheonggukjang (CGJ) is a soybean‐based quick‐fermented food popular in Korea that contains a variety of biologically active compounds including isoflavones and saponins. Isoflavone bioavailability may be important for the bone health of postmenopausal women; therefore, the aim of this study is to evaluate the influence of fermentation on the isoflavone metabolite nutrikinetic profile after single dose CGJ or unfermented soybean administration in ovariectomized (OVX) and sham mice.

2 Methods and results

We identify 34 isoflavone metabolites using UPLC–QTOF‐MS and analyze their nutrikinetics at different time points (0.25, 0.5, 1, 2, 4, 8, 16, and 24 h) to understand their fermentation‐ and OVX‐mediated time‐dependent concentration changes. Nutrikinetics analysis shows that genistein, daidzein, genistein 4′‐sulfate, dihydrodaidzein sulfate, equol 4′‐sulfate, and equol‐7‐glucuronide are present at high concentrations in all groups based on area‐under‐the‐curve analysis. OVX mice appear to show lower isoflavone bioavailability than mice in the sham group. CGJ enhances various isoflavone metabolite bioavailability including genistein, 3‐hydroxygenistein, and equol 7‐glucuronide, compared to the unfermented soybean‐treated group. Among these metabolites, intact isoflavones, 3‐hydroxygenistein, genistein 4′‐sulfate, and equol 7‐glucuronide promote osteoblastogenesis and inhibit osteoclast formation.

3 Conclusions

CGJ has good isoflavone bioavailability and may be beneficial for the bone health of postmenopausal women.     

Comparison of the in vitro metabolism of isoflavones by fecal flora from human flora‐associated mice and human

Recently there has been great interest in the biological effects of equol (4′,7‐dihydroxyisoflavan), a metabolite of daidzein produced by intestinal flora. In this study, the in vitro metabolism of daidzein, daidzin and genistein by fecal flora, in human flora‐associated (HFA) mice and a human donor was investigated. The in vitro incubation of feces with isoflavones demonstrated that the potential for equol production had been successfully transferred from human feces to HFA mice. The average equol concentrations were higher in feces from HFA mice than in those from their donor. In both HFA mice and their donor, the fecal equol production from daidzin was comparable to that from daidzein. The results suggest that daidzin would be an effective substrate for bacterial equol production in vivo. The HFA mice produced in these experiments and maintained in an isolated environment might also be useful for investigating the biological activity of equol in detail. Copyright © 2006 Society of Chemical Industry     

Aquatic photochemistry of isoflavone phytoestrogens: degradation kinetics and pathways

Isoflavones are plant-derived chemicals that are potential endocrine disruptors. Although some recent studies have detected isoflavones in natural waters, little is known about their aquatic fates. The photochemical behaviors of the isoflavones daidzein, formononetin, biochanin A, genistein, and equol were studied under simulated solar light and natural sunlight. All of these phytoestrogens were found to be photolabile under certain conditions. Daidzein and formononetin degraded primarily by direct photolysis. Their expected near-surface summer half-lives in pH 7 water at 47° latitude are expected to be 10 and 4.6 h, respectively. Biochanin A, genistein, and equol degraded relatively slowly by direct photolysis at environmentally realistic pH values, though they showed significant degradation rate enhancements in the presence of natural organic matter (NOM). The indirect photolysis rates for these compounds scaled with NOM concentration, and NOM from microbial origin was found to be a more potent photosensitizer than NOM from terrestrial sources. Mechanistic studies were performed to determine the indirect photolysis pathways responsible for the rate enhancements. Results of these studies implicate reaction with both singlet oxygen and excited state triplet NOM. Environmental half-lives for biochanin A, genistein, and equol are expected to vary on the basis of pH as well as NOM source and concentration.